Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo

Hamilton, Mark; Wolfman, Alan

doi:10.1038/sj.onc.1201653

Cited by 64 publications

(65 citation statements)

References 31 publications

(53 reference statements)

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“…Biochemical analysis revealed that Raf-1 failed to interact with the activated Ha-Ras protein in the V12H10 cells, but instead, Raf-1 was found to be associated with the wild-type endogenous c-N-Ras protein. Suramin treatment disrupted the c-NRas⅐Raf-1 complex in V12H10 cells but did not disrupt the N-Ras⅐Raf-1 complex detected in the N-Ras-transformed K61N10 cells (15). The failure of oncogenic Ha-Ras to associate with Raf-1 is contrary to current dogma, which suggests that it is the direct association of an oncogenic Ras with Raf-1 that is responsible for regulating the MAPK cascade.…”

contrasting

confidence: 52%

“…Cell Culture and Transfections-V12H10 and K61N10 fibroblasts are derived from normal mouse C3H10T1/2 fibroblasts by transfection with activated (G12V)Ha-Ras and (Q61K)N-Ras constructs, respectively and are described elsewhere (15). All cell lines were maintained in DMEM ϩ fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

“…We recently reported that mouse fibroblasts transformed by the minimal expression of activated (G12V)Ha-Ras (V12H10 cells) were unable to proliferate in serum-free medium in the presence of suramin, a growth factor antagonist (13)(14)(15). The constitutive MAPK activity in these cells was completely abrogated by the addition of suramin.…”

mentioning

confidence: 99%

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Oncogenic Ha-Ras-dependent Mitogen-activated Protein Kinase Activity Requires Signaling Through the Epidermal Growth Factor Receptor

Hamilton

Wolfman

1998

Journal of Biological Chemistry

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C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serumfree medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serumindependent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor ␣ and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.The Ras GTPases are critical transducers of signals that are initiated from plasma membrane receptor tyrosine kinases and terminate in the nucleus as changes in gene expression. The c-Ras 1 proteins are implicated in the regulation of multiple biological processes as diverse as cell proliferation, migration, and differentiation. In most instances, the expression of mutated, oncogenic forms of Ras results in cell transformation. The loss of normal growth control may result in the acquisition of anchorage-independent growth and a diminished requirement for exogenous serum or growth factors (1) and are intimately related to the ability of Ras to stimulate the MAPK cascade, a critical component of the proliferative response (2-6). Deregulated cell proliferation is thought to be one consequence of activated Ras signaling directly and persistently through the Raf kinases, upstream mediators of the MAPK cascade. Ras transformation can also be accompanied by the synthesis and secretion of various growth factors, including interleukin 1␣, interleukin 6 (7), TGF-␣ (8 -10), vascular endothelial growth factor (VEGF) (11), and heparin-binding EGF (HB-EGF) (12). The production of autocrine growth factors may therefore be a potentially significant mechanism contributing to the ability of activated Ras proteins to undermine the processes that regulate normal cell proliferation.We recently reported that mouse fibroblasts transformed by the minimal expression of activated (G12V)Ha-Ras (V12H10 cells) were unable to proliferate in serum-free medium in the presence of suramin, a growth factor antagonist (13-15). ...

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contrasting

confidence: 52%

Section: Methodsmentioning

confidence: 99%

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Oncogenic Ha-Ras-dependent Mitogen-activated Protein Kinase Activity Requires Signaling Through the Epidermal Growth Factor Receptor

Hamilton

Wolfman

1998

Journal of Biological Chemistry

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“…Similar results have been obtained using indirect antagonism of Ras with HMG-CoA Reductase inhibitors and prenylation inhibitors, which reduce the C terminal prenylation required for Ras function. Although the specific function of each of the Ras isoforms is not known, different Ras isoforms have been shown to differ in their ability to activate certain effectors (21,22), and it may be that the different isoforms of Ras play distinct roles in control of proliferation acting via different downstream cascades.…”

Section: Discussionmentioning

confidence: 99%

Ras Antagonist Farnesylthiosalicylic Acid (FTS) Reduces Glomerular Cellular Proliferation and Macrophage Number in Rat Thy-1 Nephritis

Clarke¹,

Kocher²,

Khwaja³

et al. 2003

Journal of the American Society of Nephrology

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Abstract. Targeting the Ras family of monomeric GTPases has been suggested as a therapeutic strategy in proliferative renal diseases. This article reports the effects of Ras antagonist farnesylthiosalicylic acid (FTS) in rat thy-1 nephritis, a model in which cytokine-driven glomerular cell proliferation and invasion is likely to involve Ras signaling pathways. FTS in vitro specifically inhibits the binding of Ras to discrete membrane sites, thereby downregulating several Ras-dependent signaling functions and accelerating Ras degradation. Forty-four Lewis rats were given nephritis by day zero injection of a monoclonal thy-1 antibody ER4 (2.5mg/kg body wt). Twentytwo rats were then treated with daily intraperitoneal injection of FTS (5 mg/kg body wt) until sacrifice, and the remaining control rats were given vehicle alone (C). Six rats from each group were sacrificed at day 1 to establish equal injury; other sacrifice points were day 7 and day 10. Bromo-deoxyuridine (BrdU) was injected 1 h before sacrifice, after which sections were used for immunohistochemistry, which included detection of Ras expression, BrdUϩ cells and macrophages/monocytes (ED1ϩ). Thy-1 nephritis was associated with an increase in glomerular expression of Ki-Ras and N-Ras isoforms, which was almost fully prevented by FTS. FTS treatment was associated with: (a) a 54% reduction in the mean number of BrdUϩ cells per glomerulus (P Ͻ 0.01), (b) a 50% reduction in macrophages/monocytes (ED1ϩ) per glomerulus (P Ͻ 0.01), and (c) a reduction in 24-h proteinuria at day 10 (P Ͻ 0.05). These results show that Ras inhibition can reduce both glomerular cell proliferation and glomerular macrophage cell number in the thy-1 model and justify further study of FTS as a potential therapeutic in proliferative nephritis.

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“…1). It has been recognized that activation of different Ras isoforms results in distinct signaling outputs (9,14,15,(35)(36)(37), and the membrane microdomains such as rafts and caveolae most likely contribute to the segregated signaling (11,12). It should be noted that in some cell lines established from nonneural tissues H-Ras operates in membrane rafts, whereas K-Ras seems to be located outside rafts (13,38), in contrast with the selective enrichment of K-Ras in the rafts of the rat brain membranes (Fig.…”

Section: Fig 3 Gdp and Gtp␥s Inhibit Binding Of K-ras To Scop Lrrmentioning

confidence: 99%

Suprachiasmatic Nucleus Circadian Oscillatory Protein, a Novel Binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates MAPK Pathway

Shimizu

Okada

Nonomura

et al. 2003

Journal of Biological Chemistry

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Suprachiasmatic nucleus circadian oscillatory protein (SCOP) is a member of the leucine-rich repeat (LRR)-containing protein family. In addition to circadian expression in the rat hypothalamic suprachiasmatic nucleus, SCOP is constitutively expressed in neurons throughout the rat brain. Here we found that a substantial amount of SCOP was localized in the brain membrane rafts, in which only K-Ras was abundant among Ras isoforms. SCOP interacted directly through its LRR domain with a subset of K-Ras in the guanine nucleotide-free form that was present in the raft fraction. This interaction interfered with the binding of added guanine nucleotide to K-Ras in vitro. A negative regulatory role of SCOP for K-Ras function was examined in PC12 cell lines stably overexpressing SCOP or its deletion mutants. Overexpression of full-length SCOP markedly down-regulated ERK1/ERK2 activation induced by depolarization or phorbol ester stimulation, and this inhibitory effect of overexpressed SCOP was dependent on its LRR domain. These results strongly suggest that SCOP negatively regulates K-Ras signaling in the membrane rafts, identifying a novel mechanism for regulation of the Ras-MAPK pathway.

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Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo

Cited by 64 publications

References 31 publications

Oncogenic Ha-Ras-dependent Mitogen-activated Protein Kinase Activity Requires Signaling Through the Epidermal Growth Factor Receptor

Oncogenic Ha-Ras-dependent Mitogen-activated Protein Kinase Activity Requires Signaling Through the Epidermal Growth Factor Receptor

Ras Antagonist Farnesylthiosalicylic Acid (FTS) Reduces Glomerular Cellular Proliferation and Macrophage Number in Rat Thy-1 Nephritis

Suprachiasmatic Nucleus Circadian Oscillatory Protein, a Novel Binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates MAPK Pathway

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